Two-photon thermal bleaching of single fluorescent molecules

Area of Science:

• Photophysics
• Spectroscopy
• Biophysics

Background:

• Fluorescence microscopy is crucial for bioimaging.
• Understanding dye photophysics, including photobleaching, is essential for accurate imaging.
• Two-photon excitation offers advantages in deep tissue imaging but requires specific photophysical characterization.

Purpose of the Study:

• To investigate the fluorescence emission and bleaching mechanisms of common bioimaging dyes under single-molecule, two-photon excitation.
• To differentiate between photobleaching and other quenching mechanisms.
• To correlate bleaching behavior with molecular properties and environmental factors like temperature.

Main Methods:

• Single-molecule fluorescence spectroscopy using two-photon excitation.
• Spin coating of dye molecules (rhodamine 6G, fluorescein, pyrene, indo-1) onto glass substrates.
• Systematic variation of glass substrate temperature.
• Numerical simulations to model thermal effects.

Main Results:

• Single dye molecules showed a constant fluorescence output followed by a sudden transition to a dark state.
• Bleaching times followed a Gaussian distribution, inconsistent with typical photobleaching.
• Bleaching time decreased with increasing temperature, with a temperature-dependent series (pyrene < indo-1 < fluorescein < rhodamine 6G).
• Bleaching correlated with absorbed power and molecular complexity.

Conclusions:

• The observed phenomenon is interpreted as thermal bleaching, where two-photon absorption induces localized heating.
• Temperature increase, not photobleaching, is the primary cause of the transition to the dark state.
• Molecular complexity and absorbed power influence the rate of thermal bleaching.